Dihydrofolate reductase



Dihydrofolate reductase (DHFR) is an enzyme which uses the co-factor NADPH as electron donor which converts it to NADP. It catalyzes the reduction of dihydrofolic acid to tetrahydrofolic acid (folate). The folate is a form of the essential vitamin B9. Antifolate inhibitors are used in cancer therapy. DHFR forms a complex with thymidylate synthase (TS). Both enzymes participate in the biosymthesis of pyrimidine. The images at the left and at the right correspond to one representative dihydrofolate reductase structure, i.e. the crystal structure of Moritella profunda dihydrofolate reductase (3ia5).



Structure and Function
DHFR is a ubiquitous enzyme found in all organisms. The primary physiological role of DHFR is maintenance of the intracellular levels of tetrahydrofolate, a precursor of cofactors required for the biosynthesis of purines, pyrimidines, and several amino acids.

The enzyme, which is the sole source of tetrahydrofolate catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) by stereospecific hydride transfer from the NADPH cofactor to the C6 atom of the pterin ring with concomitant protonation at N5. Being the sole source of THF it is an Achilles’ heel of rapidly proliferating cells, making it an attractive target of several important anticancer and antimicrobial drugs such as methotrexate, trimethoprim, and pyrimethamine.

E. coli DHFR is a small (18 kD) protein with an α/β structure consisting of a central eight-stranded β-sheet and four flanking α-helices. The active site cleft divides the protein into two structural subdomains: the Adenosine binding subdomain (red) and the major subdomain (green). The adenosine binding subdomain is the smaller of the two subdomains and provides the binding site for the adenosine moiety of the cofactor. The major subdomain consists of ~100 residues from the N and C termini and is dominated by a set of three loops on the ligand binding face that surround the active site. In terms of sequence length, these loops make up approximately 40%–50% of the major subdomain; hence it is sometimes called the “loop” subdomain. The loops are termed Met20, F-G, and G-H loops. Hinge bending motions about Lys38 and Val88 allow the adenosine binding domain to move relative to the major domain upon binding of various ligands, resulting in closure of the active site cleft.



A movie depicting the conformational changes of DHFR during one turnover of substrate has been constructed by M. R. Sawaya and J. Kraut using six isomorphous crystallographic structures. http://chem-faculty.ucsd.edu/kraut/dhfr.html

Additional Resources
For additional information, see: Cancer

3D Structures of Dihydrofolate reductase
Update June 2011

Native DHFR
1ohk, 1ohj – hDHFR – different crystal forms - human

3f8y, 3f8z, 3f91, 3eig, 1dlr, 1dls – hDHFR (mutant)

3ia5 – MpDHFR – Mritella profunda

2kgk, 2qk8 – BaDHFR – Bacillus anthracis

3dfr – LcDHFR – Lactobacillus casei

2w9t – SaDHFR S1 - Staphylococcus aureus

2ith, 1vdr – HvDHFR - Haloferax volcanii

2rh2, 2gqv, 1vie, 1vif, 1dfr, 5dfr – EcDHFR – Eschericia coli

1dhi, 1dhj, 2d0k – EcDHFR (mutant)

1dyr – PncDHFR - Pneumocystis carinii

1zdr – BsDHFR – Bacillus stearothermophilus

1juv – DHFR – Bacteriophage T4

1ai9 – CaDHFR - Candida albicans

1cz3, 1d1g – DHFR – Thermotoga maritima

3q1h – DHFR – Yersinia pestis

2l28 – DHFR – Lactobacillus casei - NMR

DHFR-Thymidine synthase (TS)_complex
3cl9, 2h2q – TcDHFR-TS - Trypanosoma cruzi

1qzf - ChTcDHFR-TS – Cryptosporidium hominis

3dl5, 3dl6, 2oip – ChDHFR-TS (mutant)

3i3r, 3kjr - BbDHFR-TS – Babesia bovis

3clb – TcDHFR-TS + TMX

3irm, 3iro - TcDHFR-TS + inhibitor

3irn - TcDHFR-TS+NADPH +inhibitor

3dg8, 1j3i, 3dga - PfDHFR-TS+NADPH +dUMP+inhibitor – Plasmodium falciparum

1j3k, 1j3j, 3jsu - PfDHFR-TS (mutant)+NADPH+dUMP+ inhibitor

3inv - TcDHFR-TS+NADPH+dUMP+inhibitor

3kjs - TcDHFR-TS+NADPH +inhibitor

3k2h, 3nrr - BbDHFR-TS+NADPH+dUMP+inhibitor

DHFR+antifolate inhibitors
Binary complexes

3fra – SaDHFR (mutant)+iclaprim

3fl8 – BaDHFR+RAB1

3fl9 – BaDHFR+trimethoprim

2w9h – SaDHFR+trimethoprim

2hm9 – LcDHFR+trimethoprim

1ao8 - LcDHFR+MTX – NMR 1dis, 1diu - LcDHFR+ brodimoprim – NMR 1dra, 1drb, 2drc, 3drc, 1dds, 4dfr - EcDHFR+MTX

2inq – EcDHFR+MTX – neutron diffraction

1rx5, 3och - EcDHFR+ inhibitor

2bl9, 2bla, 2blb, 2blc – PvHDFR+pyrimethamine – Plasmodium vivax

1s3u, 1s3v, 1s3w – hDHFR+tetrahydroauinazoline

1mvs, 1mvt, 1pd8, 1pd9, 1pdb, 1mvs, 1mvt, 3fs6, 1hfp, 1hfq, 1hfr, 2c2s, 2c2t, 2w3b, 3gyf, 3nxo, 3nxr, 3nxt, 3nxv – hDHFR+pyrimidine derivatives

3l3r, 3gi2, 3ghc, 3ghv, 3ghw – hDHFR (mutant)+pyrimidine  derivatives

2cig – MtDHFR+isoniazid derivative – Mycobacterium tuberculosis

Ternary complexes

3i8a, 3f0q, 3f0s, 3f0v, 3f0x, 3f0b, 3f0u, 3fq0, 3fqc, 3fqf, 3fqo, 3fqv, 3fqz – SaDHFR (mutant)+NADPH+2, 4-diamino-5-(3-(2,5-dimethoxyphenyl)prop-1-ynyl)-6-ethylpyrimidine

3frf – SaDHFR (mutant)+iclaprim+NADPH 3frb – SaDHFR (mutant)+trimethoprim (TMP)+NADP

3fre, 2w9s – SaDHFR (mutant)+TMP+NADPH 2w3v – MaDHFR +TMP+NADPH – Mycobacterium avium

2w3w – MaDHFR+NADPH+6-((2,5-diethoxyphenyl)aminomethyl)-2,4-diamino-methylpyrido(2,3-D)pyrimidine

3fy8, 3fyw – SaDHFR + AR-101+NADPH 3fy9 – SaDHFR (mutant)+ AR-102+NADPH 3fyv – SaDHFR (mutant)+ AR-102+NADPH 3fyw – SaDHFR + AR-101+NADPH 3ia4 – MpDHFR+methotrexate (MTX)+NADPH 3hbb – TcDHFR+MTX & TMTX

3jw3, 3jw5 – BaDHFR (mutant)+trimethoprim+NADPH 3dat - BaDHFR+ MTX+NADPH<BR /> 3e0b, 3jvx, 3jwc, 3jwf, 3jwk, 3jwm – BaDHFR (mutant)+NADP+ pyrimidine derivative

3lg4 - SaDHFR (mutant)+NADP+ pyrimidine derivative

2w9g - SaDHFR+trimethoprim+NADPH<BR /> 3m08 - SaDHFR+RAB1+NADP

3m09 – SaDHFR (mutant)+RAB1+NADP

1lud - LcDHFR+NADPH+ trimethoprim – NMR<BR /> 1bzf - LcDHFR+NADPH+ TMX – NMR<BR /> 3cse, 3eej, 3eek, 3eel, 3eem - CgDHFR +NADPH+ pyrimidine derivatives – Candida glabrata

3k45, 3k47 – mDHFR+E & Z-isomer+NADPH – mouse

3d80, 3d84 – mDHFR+NADPH+2,4-diamino-6-(2’-hydrosydibenz[b,f]azepin-5-yl)methylpteridine

2fzi, 2fzj - mDHFR+NADPH+ trimethoprim

1u70 – mDHFR (mutant)+MTX+NADPH<BR /> 1dre, 1rb3, 1rg7, 1rh3, 1ra3 - EcDHFR+NADP+MTX

1rx3, 3dau - EcDHFR+NADPH+MTX

3kfy - EcDHFR+NADPH + inhibitor

1ddr - EcDHFR+MTX+urea

1tdr - EcDHFR+MTX+Te

1rc4, 1dyj - EcDHFR+NADP+5,10-dideazatetrahydrofolate

1dyh - EcDHFR+NADP+dideazafolic acid

1rx4 - EcDHFR+5,10-dideazatetrahydrofolate+monophosphoadenosine 5’-diphosphoribose

1rx6 - EcDHFR+5,10-dideazatetrahydrofolate+NADPH<BR /> 1drh - EcDHFR+5]], [[10-dideazatetrahydrofolate+folate+5-deazafolate

2ano, 2anq - EcDHFR+NADPH+MS-SH08-17

1vj3 – PcHDFR+TAB+NADPH – Plasmodium carinii

1daj, 3nz6, 3nz9, 3nzb, 3nzc, 3nzd – PcDHFR + NADPH + pyrimidine derivative

2fzh – PcDHFR+NADPH+ trimethoprim

1ly3, 1s3y – PcDHFR +NAP+quinazoline derivatives

1ly4 - PcDHFR +NAP+pyrimidine derivative

2w3a – hDHFR +TMP+NADPH<BR />

1yho - hDHFR+NADPH+ trimethoprim – NMR<BR /> 1u71, 1u72 - hDHFR (mutant)+MTX+NADPH<BR /> 1boz – hDHFR (mutant)+NADPH+quinazoline derivatives

1kms, 1kmv, 3ntz, 3nxx, 3nxy – hDHFR+NADPH+ inhibitor

1m79, 1m78, 1m7a, 1ia1, 1ia2, 1ia3, 1ia4, 1aoe – CaDHFR+NADPH+quinazoline derivatives

1df7 - MtDHFR +MTX+NADPH<BR /> 1dg5 - MtDHFR + trimethoprim +NADPH<BR /> 1dg7 - MtDHFR + 4-Br-WR99210 +NADPH<BR /> 1dr1, 1dr2, 1dr3 - cDHFR+NADP+biopterin – chicken

1dr4, 1dr5, 1dr6, 1dr7 - cDHFR+NADP+biopterin+Hg

1klk – DHFR+NADPH+PT653 – rat

1sej - DHFR +NADPH+quinazoline derivative+deoxyuridine – Cryptosporidium hominis

3ix9 – DHFR (mutant) +MTX+NADPH – Streptococcus pneumoniae

3nza - PncDHFR+NADPH+ inhibitor

DHFR+ligands
3frd – SaDHFR (mutant)+folate +NADP

2w3m– hDHFR +folate+NADPH<BR /> 1drf - hDHFR +folate

1dhf, 2dhf – hDHFR+folate+5-deazofolate

1e26 - PcDHFR-TS+NADP+folate

2jyb – HvDHFR+folate

2rk1 – EcDHFR+NADP+dihydrofolate

7dfr - EcDHFR+NAP+NADP+folate

1ra8, 1rx8 – EcDHFR+folate+monophosphoadenosine 5’-diphosphoribose

1rb2, 1rx2, 1ra2, 3ql3 - EcDHFR+folate +NADP

3ql0 – EcDHFR (mutant) +folate+NADP

1rx7, 1rd7, 1re7 - EcDHFR+folate

1rf7 - EcDHFR+dihydrofolate

1dyi - EcDHFR+NADP+folic acid

1jol, 1jom - EcDHFR+folinic acid

2cd2, 3cd2, 4cd2 - PcDHFR+NADP+folate

1cd2 - PcHDFR+folate+NADPH<BR /> 2zza – MpDHFR+ folate +NADP

DHFR+co-factors
2hqp – LcDHFR+NADPH – NMR<BR /> 2rk2, 2p4t, 1ra9, 1rx9 – EcDHFR+NADP

1rx1, 1ra1 - EcDHFR+NADPH<BR /> 6dfr - EcDHFR+NAP+NADP

1dg8 - MtDHFR +NADPH<BR /> 8dfr - cDHFR +NADPH<BR />

3k74 – EcDHFR+nanobody